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Differentiation of Leucine and Isoleucine Residues in Peptides by Consecutive Reaction Mass Spectrometry Sir: Fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS) have emerged as potent techniques for peptide sequencing (1-3), but there are some remaining problems. One such problem is the differentiation of two isomeric amino acid residues, leucine and isoleucine, in peptides (1). If the peptide in question contains only a single leucine or isoleucine residue, they can be differentiated by using MS/MS spectra of the m / z 86 immonium ions corresponding to the leucine or isoleucine residue, that is, CH3CH(CH3)CH2CH=NH2+or CH3CH2CH(CH3)CH=NH2+, present in the FAB spectrum of the peptide (4). However, this approach is not useful for peptides containing both leucine and isoleucine residues because, in the FAB spectra of such peptides, the ion at m / z 86 is usually a mixture of isomeric immonium ions formed from both residues. Another approach based on FAB and tandem mass spectrometry was proposed by Biemann’s group (5,6). They used (A, - 42)’ and (A, - 28)+ ions (d-series ions) in the daughter ion spectrum of protonated molecular ions (MH+)of a peptide where A, and A, ions are sequence-specific daughter ions in which leucine or isoleucine is now C-terminal. Although this approach is applicable to the peptide containing both residues, its usefulness is limited only to the cases where A-series daughter and the diagnostic d-series ions are sufficiently strong to be recognized. The use of w-series daughter ions (side chain cleaved Cterminal ions) was also proposed (6-9).This approach is useful for the differentiation of the isomers in the peptide containing both residues as well. However, in analogy with d-series ions, w-series ions are not always observed (7). In this paper, we present a new strategy that is widely applicable for the assignment of leucine and isoleucine in peptide molecules. The new strategy is based on consecutive reaction mass spectrometry, MS” (10-14),and consists of three steps. First, the peptides (or proteins) are digested with an endopeptidase, thermolysin. This enzyme can hydrolyze the peptide bond containing the N atom of leucine or isoleucine residues. Thus, some of the peptides in the resulting digest have leucine or isoleucine as their N-terminal residues. Second, the proteolytic digest is analyzed by FAB MS and MS/MS, and the peptides having leucine or isoleucine as the N-terminal residues are assigned from the peptides in the digest. Finally, leucine and isoleucine residues in each fragment peptide are identified on the basis of the MS3 (MS/ MS/MS) spectra of each MH+ of the leucine/isoleucine terminal peptides. The usefulness of the strategy has been demonstrated in the case of the peptide human calcitonin. EXPERIMENTAL SECTION Materials. Human calcitonin,leucylalanine, and thermolysin were purchased from Sigma Chemical Co. (St. Louis, MO). Isoleucylphenylalaninewas obtained from Research Organics, Inc. (Cleveland, OH). The peptides and enzymes were used without further purification. Preparation of Samples for Mass Spectrometric Analysis. Human calcitonin was digested with thermolysin in the usual way except for the use of a volatile buffer solution, Le., 0.1 M N ethylmorpholine/acetic acid (3). The digest (3-10 nmol) in the buffer solution (1-3 pL) was mixed with 1 pL of thioglycerol followed by glycerol (1 pL), and the mixture was applied to a stainless steel FAB target and used for analysis. Measurement of Spectra. All FAB MS, MS/MS, and MS/MS/MS spectra were obtained with a JEOL JMS-HX100 tandem mass spectrometer (EBE geometry) equipped with an additional analog circuit, “MS/MS/MS interface”,which allows
manual operation of E l and B, keeping a constant B / E l ratio. The operating parameters for the measurements were as follows: fast atom beam, Xe (6 kV); accelerating voltage, 5 kV; voltage on the conversion dynode of the postacceleration detector, -20 kV. The MS/MS spectra were obtained by the use of E2 scanning. The procedure described by Cooks, Gross, and co-workers (10) was used for the MS/MS/MS acquisition. Collision gas was introduced into the first gas cell in the first field-free region (FFR) to give approximately 70% beam attenuation of the parent ion (M,’) and into the second gas cell located at the third FFR to give approximately 90% beam attenuation of the daughter ion (Md+).The resultant daughter ions of Md+ (graddaughter ions of M +: Mg+) were analyzed by E2 scanning. To improve the signa! to noise ratio of the spectra, 10-100 E2 scans (5 s/scan) were accumulated on the data system (JEOLJMS-DA5000). The total data acquisition times varied from 1 to 10 min and were dependent on the abundance of the granddaughter ions.
RESULTS AND DISCUSSION Human calcitonin is a peptide containing 32 amino acid residues, including two leucine and one isoleucine. Figure 1 shows the FAB mass spectrum of the thermolytic digest of the peptide. At a minimum, MH+ ions of 12 peptides can be recognized in the spectrum. Structures were assigned to the digest peptides as shown in the figure on the basis of the MS/MS experiments. There are four leucine/isoleucine terminal peptides, namely peptides containing residues 4-8,9-11, 27-28, and 27-32, in the assigned fragments. The protonated molecular ions of these four peptides, i.e., m / z 554,290,189, and 512, are seen in the FAB mass spectrum (Figure 1). The mass of m / z 189 also corresponds to residues 9-10 (Leu-Gly), but the substrate specificity of the enzyme (15)suggests that the ion is the MH+ of the peptide containing residues 27-28 (Ile-Gly). Collisional activation of these four ions gave abundant N-terminal immonium ions, m / z 86. Hence, we tried to use daughter ion spectra of the immonium ions, granddaughter ion spectra (MS/MS/MS spectra) of each leucine/isoleucine terminal peptide, for the differentiation of those residues. Namely, MH+ of each fragment peptide was used as MP+,the m / z 86 immonium ion formed from M,+ in the first FFR was used as Md+, and the third FFR daughters of Md+, M,+, were analyzed by using E2. First, we looked at the MS/MS/MS spectra of simple dipeptides containing leucine and isoleucine. Figure 2 shows the MS/MS/MS spectra, the daughter ion spectra of the immonium ions at m/z 86 (Md+)formed in the first FFR from the MH+ of leucylalanine and isoleucylphenylalanine (Mp+, ionized by FAB). Both of the spectra have the same features as that of the daughter ion spectra of m / z 86 immonium ions formed from leucine and isoleucine in an ion source ( 4 ) . Higher mass peaks ( m / z 56,69) are relatively abundant for m/z 86 from isoleucylphenylalanine (Figure Zb), whereas m / z 43 and 44 are predominant in the spectrum of the immonium ion from leucylalanine (Figure 2a). Consequently, the MS/ MS/MS spectra can also be used as fingerprints of the ions in a manner similar to the case of MS/MS spectra (2,16,17). Recently, Tomer and co-workers reported that the spectra obtained by MS4 (MS/MS/MS/MS) experiments on the m / z 86 ions from Leu-Gly-Gly, Gly-Gly-Leu, and Gly-Gly-Ile were comparable with the MSjMS spectra of m / z 86 from leucine and isoleucine (14). Although their method clearly has the potential to differentiate between the isomeric amino acid residues in peptides, the reported sensitivity of the experiments (acquisition time for an MS/MS/MS/MS spectrum
0 1990 American Chemical Society 0003-2700/90/0362-0311$02.50/0
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